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J. Am. Chem. Soc. 1998, 120, 13252-13253
LNA (Locked Nucleic Acid): An RNA Mimic Forming Exceedingly Stable LNA:LNA Duplexes Alexei A. Koshkin,† Poul Nielsen,‡ Michael Meldgaard,† Vivek K. Rajwanshi,† Sanjay K. Singh,† and Jesper Wengel*,† Center for Synthetic Bioorganic Chemistry Department of Chemistry, UniVersity of Copenhagen UniVersitetsparken 5 DK-2100 Copenhagen, Denmark Department of Chemistry, Odense UniVersity DK-5230 Odense M, Denmark ReceiVed June 30, 1998 In the search for nucleic acid mimics capable of binding strongly to complementary DNA and/or RNA, synthesis of conformationally restricted oligonucleotide (ON) analogues has been intensified recently.1 Among others, ONs containing monomeric nucleotides with bicyclic and tricyclic carbohydrate moieties have been examined.2 We have recently introduced the novel ON analogue LNA (Locked Nucleic Acid; Figure 1)3-5 containing 2′-O,4′-C-methylene linked bicyclic ribofuranosyl nucleosides locked in an N-type [3′-endo/3E] conformation. In this paper, LNA:LNA hybridization is introduced as the most thermally stable nucleic acid type duplex system yet discovered, and the RNAmimicking character of LNA is established at the duplex level. Several examples of the formation of very stable nucleic acid type duplexes resulting from the pairing of two chemically modified ON strands have been described in the literature, e.g., involving pyranosyl-RNA,6,7 1′,5′-anhydrohexitol nucleic acid (HNA),7,8 bicyclo-DNA,2a,2b,7 and tricyclo-DNA.2e,7 Stimulated by the unprecedented thermal stabilities and the very satisfactory base-pairing selectivities observed for duplexes between LNA and unmodified DNA or RNA,3 we decided to evaluate the stability of LNA:LNA duplexes.9 The results from thermal denaturation studies are depicted in Table 1. From entries 1, 3, and 4 it can be extracted that introduction of three LNA monomers (TL or AL) induces significantly increased melting temperatures (∆Tm ) +15 †
University of Copenhagen. Odense University. (1) (a) Herdewijn, P. Liebigs Ann. 1996, 1337. (b) Cool, E. T. Chem. ReV. 1997, 97, 1473. (2) (a) Tarko¨y, M.; Leumann, C. Angew. Chem., Int. Ed. Engl. 1993, 32, 1432. (b) Bolli, M.; Litten, J. C.; Schu¨tz, R.; Leumann, C. J. Chem. Biol. 1996, 3, 197. (c) Altmann, K.-H.; Kesselring, R.; Francotte, E., Rihs, G. Tetrahedron Lett. 1994, 35, 2331. (d) Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.; Wang, J.; Wagner, R. W.; Matteucci, M. D. J. Med. Chem. 1996, 39, 3739. (e) Steffens, R.; Leumann, C. J. J. Am. Chem. Soc. 1997, 119, 11548. (f) Nielsen, P.; Pfundheller, H. M.; Wengel, J. Chem. Commun. 1997, 825. (g) Nielsen, P.; Pfundheller, H. M.: Olsen, C. E.; Wengel, J. J. Chem. Soc., Perkin Trans. 1 1997, 3423. (h) Christensen, N. K.; Petersen, M.; Nielsen, P.; Jacobsen, J. P.; Olsen, C. E.; Wengel, J. J. Am. Chem. Soc. 1998, 120, 5458. (3) (a) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem. Commun. 1998, 455. (b) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54, 3607. (c) Koshkin, A. A.; Rajwanshi, V. K.; Wengel, J. Tetrahedron Lett. 1998, 39, 4381. (d) Singh, S. K.; Wengel, J. Chem. Commun. 1998, 1247. (4) LNA is defined as an ON containing one or more 2′-O, 4′-C-methylene bicyclic ribonucleoside LNA monomers. (5) Synthesis of the uracil and cytosine LNA nucleosides by a linear route from uridine has been published. Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T. Tetrahedron Lett. 1997, 38, 8735. (6) (a) Pitsch, S.; Wendeborn, S.; Jaun, B.; Eschenmoser, A. HelV. Chim. Acta 1993, 76, 2161. (b) Pitsch, S.; Krishnamurthy, R.; Bolli, M.; Wendeborn, S.; Holzner, A.; Minton, M.; Lesueur, C.; Shlo¨nvogt, I.; Jaun, B.; Eschenmoser, A. HelV. Chim. Acta 1995, 78, 1621. (7) These modified duplexes all displayed increased thermal stability compared to the corresponding unmodified duplexes. (8) (a) Hendrix, C.; Rosemeyer, H.; Verheggen, I.; Seela, F.; Van Aerschot, A.; Herdewijn, P. Chem. Eur. J. 1997, 3, 110. (b) Hendrix, C.; Rosemeyer, H.; De Bouvere, B.; Van Aerschot, A.; Seela, F.; Herdewijn, P. Chem. Eur. J. 1997, 3, 1513. (9) The LNAs were synthesized and analyzed as described earlier.3b ‡
Figure 1. Structure of RNA and LNA (base ) nucleobase) and characteristics of LNA. Also shown is the locked N-type conformation of an LNA nucleoside. ∆Tm/LNA monomer ) change in melting temperature (Tm value) per LNA monomer incorporated compared to the Tm value of the corresponding unmodified duplex.
°C/+11 °C) toward the DNA complements. Remarkably, the duplex formed between the two complementary deoxy-LNAs10 each containing three LNA monomers (Table 1, entry 6; three TL:AL base pairs) exhibited a ∆Tm value of +34 °C indicating a more than additive effect of LNA:LNA base pairing on the thermal stability (entries 3, 4, and 6). An even more dramatic stabilizing effect can be induced by complex formation between ribo-LNA10 and deoxy-LNA. Thus, an increased thermal stability corresponding to a ∆Tm value of +47 °C was obtained (Table 1, entry 7 compared to entry 2; three TL:AL base pairs) reflecting the pronounced effect of LNA monomers on the base-pairing properties of oligoribonucleotides.3d As the next step, all-modified LNAs were evaluated. In line with the results described above, a more than additive increase in the thermal stability (∆Tm ) + 56 °C) of the duplex between an all-modified LNA and the deoxy-LNA containing three AL monomers was observed (Table 1, entry 8 compared with entries 1, 4, and 5). Furthermore, LNA:LNA complex formation between two all-modified LNAs was evaluated (Table 1, entries 9 and 10). For the complementary LNAs (entry 9) we were unable to detect a duplex dissociation within the range of measurement in the standard medium salt buffer (Tm estimated >93 °C). However, in a low salt buffer (1 mM sodium phosphate) a transition (∆Tm ∼93 °C) was evident. It is noteworthy that we were unable to detect a transition for the corresponding reference DNA:DNA duplex when using the low salt conditions (Table 1, entry 1; Tm estimated +64d/>+83e
>93d/76e
>+64d/>+66e
a A ) adenosine monomer, C ) cytidine monomer, G ) guanosine monomer, U ) uridine monomer, T ) thymidine monomer, MeC ) 5-methylcytidine monomer, XL ) LNA monomer. Oligo-2′-deoxynucleotide sequences are depicted as d(sequence) and oligoribonucleotide sequences as r(sequence). b The melting temperatures (Tm values) were obtained as the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded as described earlier3b using 1.5 µM concentrations of the two complementary strands (assuming identical extinction coefficients for LNA and the corresponding unmodified strands). All transitions were monophasic. c ∆Tm values are the overall increases in the thermal stability compared to the corresponding reference duplex. d Medium salt buffer: 10 mM sodium phosphate, pH 7.0, 100 mM sodium chloride. e Low salt buffer: 1 mM sodium phosphate, pH 7.0. f Measured in medium salt buffer containing EDTA: 10 mM sodium phosphate, pH 7.0, 100 mM sodium chloride, 0.1 mM EDTA. In our experience, the effect of the added EDTA on the Tm values is within (1 °C. g Calculated relative to entry 2; All other ∆Tm values are calculated relative to entry 1.
Table 2. Thermodynamic Dataa duplex
-∆H (kJ/mol)
-∆S (kJ/(mol K))
-∆G37 °C (kJ/mol)
Table 1; entry 1 Table 1; entry 2 Table 1; entry 3 Table 1; entry 5
317 287 294 423
0.94 0.85 0.81 1.13
27 25 42 73
a Determined from the linear relations between 1/T 14 M and ln[c]. Measured in medium salt buffer: 10 mM sodium phosphate, pH 7.0, 100 mM sodium chloride. Duplex concentration c ) 1.5-35 µM (see footnote to Table 1 for further details).
general applicability of high-affinity nucleic acid analogues in supramolecular chemistry. CD spectra of different complexes involving all-modified LNA are depicted as Supporting Information together with a CD spectrum of the corresponding RNA:RNA duplex. As anticipated from the restriction of LNA monomers into an N-type conformation,3 the CD spectra show that the LNA:DNA, LNA:RNA, and LNA:LNA complexes all structurally resemble the A-form duplex of the RNA:RNA reference,1a,13 Especially the LNA:RNA complex appears to be an excellent mimic of the reference RNA: RNA A-form duplex. The structure of the LNA:DNA and LNA: LNA complexes deviate to some extent from the structure of the RNA:RNA duplex, and elements in the CD spectrum characteristic of a B-form duplex are apparent. Preliminary thermodynamic evaluation of LNA-mediated nucleic acid recognition has been performed from the concentration dependencies of Tm (Table 2).14 As expected from the strong preorganization of LNA, the introduction of three LNA monomers (TL) (Table 1; entry 3) seems to induce an entropically favored duplex formation toward DNA, compared to the unmodified (12) A pentameric guanidinium-linked thymidylate has been reported to display an estimated Tm value of more than 100 °C towards poly(rA). However, it has been shown that this association involves a triple helical complex. (a) Brown, K. A.; Dempcy, R. O.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6097. (b) Linkletter, B. A.; Bruice, T. C. Bioorg. Med. Chem. Lett. 1998, 8, 1285. (13) The conformational flexibility of the unmodified monomeric nucleotides when present in a double helix is strongly restricted into either an S-type conformation (2′-endo conformation, B-form duplexes) or an N-type conformation (3′-endo conformation, A-form duplexes). DNA:DNA hybrids adopt either A- or B-form whereas RNA:RNA hybrids exclusively adopt A-form as do, generally, RNA:DNA duplexes.1a (14) Marky, L. A.; Breslauer, K. J. Biopolymers 1987, 26, 1601.
duplexes (Table 1; entries 1 and 2).15 However, formation of the duplex between the all-modified LNA and complementary DNA (Table 1; entry 5) appears to be entropically disfavored and strongly enthalpically favored. The low melting temperature (
